To develop a protocol for cryopreservation and recovery of human endometrial epithelial cells (eECs) retaining molecular and functional characteristics of endometrial epithelium in vivo.In vitro study using human endometrial cells.University research laboratory.Endometrial biopsies were obtained from premenopausal women undergoing benign gynecologic procedures.Primary eECs were cryopreserved in 1% fetal bovine serum/10% dimethylsulfoxide in Defined Keratinocyte Serum-Free Medium (KSFM). Recovered cells were observed for endometrial stromal fibroblast (eSF) contamination and subsequently evaluated for morphology, gene expression, and functional characteristics of freshly cultured eECs and in vivo endometrial epithelium.Analysis of eEC morphology and the absence of eSF contamination; evaluation of epithelial-specific gene and protein expression; assessment of epithelial polarity.Endometrial epithelial cells recovered after cryopreservation (n = 5) displayed epithelial morphology and expressed E-cadherin (CDH1), occludin (OCLN), claudin1 (CLDN1), and keratin18 (KRT18). Compared with eSF, recovered eECs displayed increased (P<.05) expression of epithelial-specific genes AREG, CDH1, DEFB4A, MMP7, and WNT7A, while exhibiting low-to-undetectable (P<.05) stromal-specific genes COL6A3, HOXA11, MMP2, PDGFRB, and WNT5A. Recovered eECs secreted levels of cytokines and growth factors similarly to freshly cultured eECs. Recovered eECs could form a polarized monolayer with high transepithelial electrical resistance (TER) and impermeability to small molecules, and expressed apical/basolateral localization of CDH1 and apical localization of OCLN.We have developed a protocol for cryopreservation of eECs in which recovered cells after thawing demonstrate morphologic, transcriptomic, and functional characteristics of human endometrial epithelium in vivo.